Wound dressing and method for controlling severe, life-threatening bleeding

ABSTRACT

This invention is directed to advanced hemorrhage control wound dressings, and methods of using a producing same. The subject wound dressing is constructed from a non-mammalian material for control of severe bleeding. The wound dressing is formed of a biomaterial comprising chitosan for controlling severe bleeding. The kind of severe, life-threatening bleeding contemplated by this invention is typically of the type not capable of being stanched when a conventional gauze wound dressing is applied with conventional pressure to the subject wound. The wound dressing being capable of substantially stanching the flow of the severe life-threatening bleeding from the wound by adhering to the wound site, to seal the wound, to accelerate blood clot formation at the wound site, to reinforce clot information at the wound site and prevent bleed out from the wound site, and to substantially prohibit the flow of blood out of the wound site.

This application claims priority under 35 U.S.C. § 119 ProvisionalApplication No. 60/298,773, filed in the United States on Jun. 14, 2001,the entire contents of which are hereby incorporated by reference.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to license others on reasonable terms asprovided by the terms of Grant No. DAMD17-98-1-8654 awarded by theArmy/MRMC—Medical Research and Material Command.

BACKGROUND OF THE INVENTION

An advanced hemorrhage control bandage and methods of its applicationwould substantially augment available hemostatic methods. To date, theapplication of continuous pressure with gauze bandage remains thepreferred primary intervention technique used to stem blood flow,especially that from severely bleeding wounds. However, this procedureneither effectively nor safely stanches severe blood flow. This hasbeen, and continues to be, a major survival problem in the case ofsevere life-threatening bleeding from a wound.

Furthermore, it is widely accepted that severe bleeding is the leadingcause of death from wounds on the battlefield, accounting forapproximately 50 percent of such deaths. It is estimated that one-thirdof these deaths may be preventable with enhanced hemorrhage controlmethods and devices. Such enhanced hemorrhage control would also provemost useful in the civilian population where hemorrhage is the secondleading cause of death following trauma.

Currently available hemostatic bandages, restricted to use in surgicalapplications, such as collagen wound dressings or dry fibrin thrombinwound dressings are not sufficiently resistant to dissolution in highblood flow nor do they have strong enough adhesive properties to serveany practical purpose in the stanching of severe blood flow. Thesecurrently available surgical hemostatic bandages are also delicate andthus prone to failure should they be damaged by bending or loading withpressure.

There is prior art relating to chitosan and chitosan dressings. Forexample, U.S. Pat. No. 4,394,373 employs chitosan in liquid or powderform to agglutinate blood in microgram/ml quantities. Also, U.S. Pat.No. 4,452,785 is directed to a method of occluding blood vesselstherapeutically by injecting chitosan directly into the vessels. U.S.Pat. No. 4,532,134 further relates to hemostatis, inhibitingfibroplasias, and promoting tissue regeneration by placing in contactwith the tissue wound a chitosan solution or water-soluble chitosan. Thechitosan forms a coagulum which prevents bleeding.

Moreover, U.S. Pat. No. 5,858,350 relates to a process to make diatomderived biomedical grade, high purity chitin and chitin derivatives (socalled protein-free even though this is not demonstrated by analysis inthe patent). The proposed advantage of so called protein-freechitin/chitosan materials are that they should be significantly lessantigenic than current shrimp and crab derived chitin materials.

Mi, F L, et al, Fabrication and Characterization of a Sponge-LikeAssymetric Chitosan Membrane as a Wound Dressing, Biomaterials,22(2):165-173 (2001) describes the fabrication and wound healingfunction of an asymmetric chitosan membrane produced by a phaseinversion method.

Chan, M W, et al, Comparison of Poly-N-acetyl Glucosamine (P-GlcNAc)with Absorbable Collagen (Actifoam), and Fibrin Sealant (Bolheal) forAchieving Hemostasis in a Swine Model of Splenic Hemorrhage, J. Trauma,Injury, Infection, and Critical Care, 48(3):454-458 (2000) describes thetesting of chitin/chitosan hemostatic patches under the moderate bloodflow and oozing typical of the swine spleen capsular stripping test.

Cole, D. J., et al, A Pilot Study Evaluating the Efficacy of a FullyAcetylated poly-N-acetyl glucosamine Membrane Formulation as a TopicalHemostatic Agent, Surgery 126(3):510:517 (1999) describes hemostaticagent testing in the swine spleen capsular stripping test.

Sandford, Steinnes A., “Biomedical Applications of High Purity Chitosan”in Water Soluble Polymers, Synthesis, Solution Properties andApplications, ACS Series 467, Shalaby W S. McCormick C L. Butler G B.Eds. ACS, Washington, D.C. 1991, Ch 28, 431-445. This is a generalreview paper describing chitosan use with reference to a chitosansponge.

Mallette, W. G., et al, Chitosan: A New Hemostat, The Annals of ThoracicSurgery, 36(1), 55-58, (1983) See comments concerning the Malettepatents above.

Olsen, R., et al, In Chitin and Chitosan, Sources, Chemistry,Biochemistry, Physical Properties and Applications, Elsevier AppliedScience, London and New York, 1989, 813-828. This paper concerns theagglutinating efficiency of chitosan.

Japanese Patent 60142927 covers a chitosan medical band with improvedtack Japanese patent 63090507 A2 describes a water insoluble and 2%acetic acid insoluble chitosan sponge for external hemostaticapplication or for protection of a wound.

U.S. Pat. No. 5,700,476 describes collagen based structurallyinhomogeneous sponges for wound dressings and/or implant applicationsformed by freeze drying techniques employing at least onepharmacological agent and at least one substructure.

U.S. Pat. No. 2,610,625 relates to freeze dried sponge structures thatare highly effective in stopping the flow of blood or other fluids andwhich will be absorbed after a time in the body. This patent describescollagen sponge preparation.

U.S. Pat. No. 5,836,970 comprises a wound dressing formed of a blend ormixture of chitosan and alginate.

SUMMARY OF THE INVENTION

The invention is directed to a first-aid/primary intervention wounddressing for control of severe, life-threatenincg bleeding. The subjectwound dressing is typically relatively low cost. Presently there are nolow cost wound dressings that address or any wound dressings that aresuitable for control of severe life-threatening bleeding. Such bleedingcan be fatal in ballistic injuries and severe arterial lacerations.There is an urgent need for this type of dressing especially in thebattlefield where typically 50% of all deaths are associated with aninability to immediately control severe bleeding.

An advanced wound dressing for control of severe, life-threateningbleeding should preferably have the following properties:

i) easily and quickly applied in one step after removal from package

ii) rapid and strong blood clotting

iii) rapid and strong tissue adhesion

iv) strong internal cohesive properties

v) rapid and strong wound sealing

vi) resistant to dissolution under strong blood flow

vii) able to be treated roughly without compromising efficacy

This invention is directed to advanced hemorrhage control wounddressings, and methods of using and producing same. The subject wounddressing is constructed from a non-mammalian material for control ofsevere bleeding. The preferred non-mammalian material is poly[β-(1→4)-2-amino-2-deoxy-D-glucopyranose ]more commonly referred to aschitosan.

In general, the subject dressing is formed of a biomaterial comprisingchitosan for controlling severe bleeding. Preferably, the biomaterialcomprises a non-mammalian material. The kind of severe, life-threateningbleeding contemplated by this invention is typically of the type notcapable of being stanched when a conventional gauze wound dressing isapplied with conventional pressure to the subject wound. Alternatively,the nature of the severe, life-threatening bleeding is such that it isnot capable of being stanched when a conventional gauze wound dressingis applied with conventional pressure to the wound and, if notcontrolled by other means, would result in the person lapsing into astate of hypotension. Stated another way, the severe, life-threateningbleeding is generally not capable of being stanched when a conventionalgauze wound dressing is applied with conventional pressure to the woundand, if not controlled by other means, would result in the systolicblood pressure of the person dropping to a level of less than about 90mm Hg.

The severe, life-threatening bleeding can also be described as a steadyhigh flow of blood of more than about 90 ml of blood loss per minute,such that in about 20 minutes of bleeding a volume of more than about40% of total blood from a 70 kg human male would be lost, and the bloodvolume loss would substantially reduce the likelihood of survival of theperson. In many cases, the severe bleeding is caused by a ballisticprojectile injury or a sharp perforation injury or a blunt traumaticinjury. In other cases, the severe bleeding is caused by coagulopathy orinternal trauma or surgical trauma.

The wound dressing is preferably capable of stanching said severebleeding which is caused by a substantial arterial wound or asubstantial venous wound having a blood flow rate of at least about 90ml/minute. The wound dressing is also preferably capable of adhering tothe wound site by the application of direct pressure to the wounddressing for a period of time of not more than about five minutes. Thewound dressing also preferably acts quickly to seal the wound. The wounddressing also preferably facilitates substantial clotting andagglutinating of the severe bleeding from the wound site, and stanchesthe severe bleeding with the temporary application of direct pressure tothe wound dressing. The wound dressing preferably has a high resistanceto dissolution in high blood flow. The wound dressing preferably hasgood internal cohesion properties and thus has sufficient flexibilityand toughness to resist rough handling.

The wound dressing is typically produced from a chitosan biomaterial andformed into a sponge-like or woven configuration via the use of anintermediate structure or form producing steps. Such structure or formproducing steps are typically carried out from solution and can beaccomplished employing techniques such as freezing (to cause phaseseparation), non-solvent die extrusion (to produce a filament),electro-spinning (to produce a filament), phase inversion andprecipitation with a non-solvent (as is typically used to producedialysis and filter membranes) or solution coating onto a preformedsponge-like or woven product. In the case of freezing, where two or moredistinct phases are formed by freezing (typically water freezing intoice with differentiation of the chitosan biomaterial into a separatesolid phase), another step is required to remove the frozen solvent(typically ice), and hence produce the wound dressing without disturbingthe frozen structure. This can be accomplished by a freeze-drying and/ora freeze substitution step. The filament can be formed into a non-wovensponge-like mesh by non-woven spinning processes. Alternately, thefilament can be produced into a felted weave by conventional spinningand weaving processes. Other processes that may be used to make the saidbiomaterial sponge-like product include dissolution of added porogensfrom a solid chitosan matrix or boring of material from said matrix.

The wound dressing is preferably formed of a biomaterial comprising aninterconnected open porous structure, and/or an oriented open lamellastructure, and/or an open tubular structure, and/or an open honeycombstructure, and/or a filamentous structure. The wound dressing hasinterconnected free-space domains or pores with pore diameters ofpreferably at least about 15 microns, more preferably at least about 30microns, most preferably at least about 35 microns, preferably up toabout 100 microns, more preferably up to about 125 microns, and mostpreferably up to about 150 microns.

The wound dressing has an available blood contacting surface area perbase surface of said wound dressing of preferably at least about 100 cm²per cm², more preferably at least about 200 cm² per gram per cm², andmost preferably at least about 300 cm² per gram per cm². The availablemass of chitosan biomaterial per wound surface area is preferably atleast about 0.02 g/cm², more preferably at least about 0.04 g/cm², andmost preferably at least about 0.06 g/cm².

Furthermore, the wound dressing has a mean rate of dissolution per basesurface area of said wound dressing when adhered to said wound site, ata temperature of about 37° C., of preferably not more than about 0.008grams per minute per cm², more preferably not more than about 0.005grams per minute per cm², and most preferably not more than about 0.002grams per minute per cm².

The subject wound dressing preferably has a density of at least about0.05 g/cm³, more preferably at least about 0.07 g/cm³, and mostpreferably at least about 0.11 g/cm³. It can have a compression loadingpreferably to a compression density at least about 0.05 g/cm³, morepreferably at least about 0.07 g/cm³, most preferably at least about0.095 g/cm³, and preferably of not more than about 0.14 g/cm³, morepreferably not more than about 0.12 g/cm³, most preferably not more thanabout 0.10 g/cm³.

A wound dressing of this invention typically contains chitosan withnumber average molecular weight of at least about 50 kda, preferably atleast about 75 kda, more preferably at least about 100 kda, and mostpreferably at least about 150 kda (molecular weights determined by GelPermeation Chromatography relative to polyethylene glycol standards inpH 5.5, 0.01 M sodium acetate). The chitosan also preferably has aweight average molecular weight of at least about 100 kda, morepreferably at least about 150 kda, and most preferably at least about300 kda (molecular weights determined by Gel Permeation Chromatographyrelative to polyethylene glycol standards in pH 5.5, 0.01 M sodiumacetate). The chitosan in the wound dressing also has a Brookfield LVDV-II+ viscosity at 25° C. in 1% solution and 1% acetic acid (AA) withspindle LV1 at 30 rpm which is preferably not less than 100 centipoise,more preferably not less than 125 centipoise, most preferably not lessthan 150 centipoise, The molecular weights and viscosities referred toimmediately above are in respect to substantially pure chitosan wounddressings and wound dressings formed with an adsorbed surface layer ofchitosan. In the case of a wound dressing containing a covalently boundsurface layer of chitosan, then lower viscosities and molecular weightsof chitosan may be preferred.

The wound dressing of the present invention can comprise cationicchitosan salts for promoting tissue adhesion and tissue sealing.Preferably, the cationic chitosan salts are selected from a groupconsisting of chitosan formate, chitosan acetate, chitosan lactate,chitosan ascorbate and chitosan citrate. The chitosan has a degree ofdeacetylation which is typically at least about 70%, preferably at leastabout 75%, more preferably at least about 80%, most preferably at leastabout 85%.

In a preferred form of this invention, the wound dressing has a backingsupport layer attached thereto that provides for and that facilitatesimproved handling and mechanical properties. This backing layer can beattached or bonded to the dressing by direct adhesion with the top layerof chitosan, or an adhesive such as 3M 9942 acrylate skin adhesive, orfibrin glue or cyanoacrylate glue can be employed. This backing supportlayer is also preferably substantially blood insoluble. The backingsupport layer is also preferably substantially blood impermeable. Thebacking support layer is also preferably substantially biodegradable.The backing support layer is preferably a material which allows for firmhandling of the bandage during application and non-sticking to handsonce bandage has been applied.

Preferably, the material which forms the backing support layer is alayer of polymeric material. Examples of preferred backing materialsinclude low-modulus meshes and/or films and/or weaves of synthetic andnaturally occurring polymers. Synthetic biodegradable materials includepoly(glycolic acid), poly(lactic acid), poly(e-caprolactone),poly(β-hydroxybutyric acid), poly(β-hydroxyvaleric acid), polydioxanone,poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid),polyphosphazene and the copolymers of the monomers used to synthesizethe above-mentioned polymers. Naturally occurring biodegradable polymersinclude chitin, algin, starch, dextran, collagen and albumen.Non-biodegradable polymers for temporary external wound applicationsinclude polyethylene, polypropylene, metallocene polymers,polyurethanes, polyvinylchloride polymers, polyesters and polyamides.

The wound dressing of this invention has the degree of adhesion to thewound site which is preferably at least about 40 kPa, more preferably atleast about 60 kPa, and most preferably at least about 100 kPa. Also,the wound dressing has a thickness which is preferably not less thanabout 3.0 mm, more preferably not less than about 3.5 mm, and mostpreferably not less than about 4.0 mm, and preferably not more thanabout 8.0 mm, more preferably not more than about 7.0 mm, and mostpreferably not more than about 6.5 mm.

A wound dressing (2.5 cm wide) of this invention preferably has anultimate tensile breaking load of not less than 1 kg, more preferably atleast 1.5 kg and most preferably at least 2.25 kg. This same dressingpreferably has an ultimate elongation of at least 70%, more preferablyat least 90% and most preferably at least 110%. The young's modulus ofthis dressing is preferably less than 5 MPa, more preferably less than 3MPa and most preferably less than 1 MPa.

The wound dressing preferably includes a supplemental traction surfacewhich is particularly useful for the application of the wound dressingto a wound site which includes a significant amount of surface blood.The supplemental traction surface can comprise at least one outersurface which grips the wound site to avoid slipping of wound dressing,typically in a direction away from the wound site, during use. Thesupplemental traction surface is preferably in the form of a treaddesign.

The subject wound dressing is preferably capable of forming an adhesivematerial in combination with blood flowing from said wound at the wounddressing-blood interface. In this case, the adhesive material preferablyhas a pH of not more than about 5.5, more preferably not more than about4.5, more preferably not more than about 4, when the wound is sealed.Typical acids employed for purposes of adjusting the pH of the wounddressing are as follows: acetic acid, formic acid, lactic acid, ascorbicacid, hydrochloric acid and citric acid. The mole ratio of acid anion toglucosamine functional groups in the chitosan cation/anion pair toadjust the pH to the level described above is preferably about 0.90,more preferably about 0.75, and most preferably about 0.60.

The wound dressing is preferably capable of being conformed to theconfiguration of the wound, for engagingly contacting the wound, and forfacilitating stanching of the flow of the severe life-threateningbleeding. More particularly, the wound dressing is introduced into theinterstices of the wound. More preferably, the wound dressing is capableof being conformed into a tubular configuration. Then, the reconfiguredwound dressing is inserted into the wound.

This invention also contemplates a method for controlling severe,life-threatening bleeding from a wound at a wound site of a person. Themethod comprises providing a wound dressing formed of a biomaterialcomprising chitosan, adhering said wound dressing to the wound site andsubstantially stanching the flow of said severe life-threateningbleeding from said wound. Preferably, the wound is sealed and bleed outis prevented from said wound site. Also, bleeding and the flow of otherfluids into and/or out of the said wound site are preferably prevented.

It has been found that the dressing typically acts to rapidly produce astrong clot at the bleeding site by agglutinating red blood cels. It canalso promote clotting by accelerating the normal platelet clottingpathway.

A method can also be provided for producing a wound dressing capable ofcontrolling severe, life-threatening bleeding from a wound at a woundsite of a person. Such a method comprises the steps of providing achitosan biomaterial as described above.

Preferably, the chitosan biomaterial is degassed. Typically, degassingis removing sufficient residual gas from the chitosan biomaterial sothat, on undergoing a subsequent freezing operation, the gas does notescape and form unwanted voids or trapped gas bubbles in the subjectwound dressing product. The degassing step can be performed by heating achitosan biomaterial, typically in the form of a solution, and thenapplying a vacuum thereto. For example, degassing can be performed byheating a chitosan solution to 60° C. immediately prior to applyingvacuum at 500 mTorr for 5 minutes while agitating the solution.

Next, the chitosan biomaterial, which is typically in solution form, issubjected to a freezing step. Freezing is preferably carried out bycooling the chitosan biomaterial solution and lowering the solutiontemperature from room temperature to a final temperature below thefreezing point. In this way, the preferred structure of thewound-dressing product can be prepared. The final freezing temperatureis preferably not more than about −10° C., more preferably not more thanabout −20° C., and most preferably not more than about −30° C.Preferably, the temperature is gradually lowered over a predeterminedtime period. For example, the freezing temperature of a chitosanbiomaterial solution can be lowered from room temperature to −45° C. byapplication of a constant temperature cooling ramp of between −0.4°C./min to −0.8° C./min for a period of 90 minutes to 160 minutes.

Preferably, the frozen chitosan biomaterial then undergoes water removalfrom within the interstices of the frozen material. This water removalstep can be achieved without damaging the structural integrity of thefrozen chitosan biomaterial. Typically, this is achieved withoutproducing a substantial liquid phase which can disrupt the structuralarrangement of the ultimate wound dressing. Thus, preferably, thechitosan biomaterial passes from a solid frozen phase into a gas phasewithout the substantial formation of an intermediate liquid phase.

The preferred manner of implementing water removal is by employing afreeze-drying step. Freeze-drying of the frozen chitosan biomaterial canbe conducted by further freezing the frozen chitosan biomaterial.Typically, a vacuum is then applied thereto. Next, it is preferred toheat the evacuated frozen chitosan material. Then, there can be apreferred step of drying the heated, evacuated, frozen chitosanmaterial.

More specifically, the frozen chitosan biomaterial can be subjected tosubsequent freezing preferably at about −15° C., more preferably atabout −25° C., and most preferably at about −45° C., for a preferredtime period of at least about 1 hour, more preferably at least about 2hour, and most preferably at least about 3 hour. This can be followed bycooling of the condenser to a temperature of less than about −45° C.,more preferably at about −60° C., and most preferably at about −85° C.Next, a vacuum in the amount of preferably at most about 150 mTorr, morepreferably at most about 100 mTorr, and most preferably at least about50 mTorr, can be applied. Then, the evacuated frozen chitosan materialcan be heated preferably at about −25° C., more preferably at about −15°C., and most preferably at about −10° C., for a preferred time period ofat least about 1 hour, more preferably at least about 5 hour, and mostpreferably at least about 10 hour. Finally drying can be conducted atpreferably at a temperature of about 20° C., more preferably at about15° C., and most preferably at about 10° C., for a preferred time periodof at least about 36 hour, more preferably at least about 42 hour, andmost preferably at least about 48 hour.

Subsequently, the chitosan biomaterial as previously treated can becompressed, such as by using heated platens, to reduce the thickness andincrease the density of said wound dressing. The compression temperatureis preferably not less than 60° C., more preferably it is not less than75° C. and not more than 85° C. Then, the pressed chitosan biomaterialis preferably preconditioned by heating same to a temperature ofpreferably up to about 75° C., more preferably to a temperature of up toabout 80° C., and most preferably to a temperature of preferably up toabout 85° C. Preconditioning is typically conducted for a period of timeup to about 0.25 hours, preferably up to about 0.35 hours, morepreferably up to about 0.45 hours, and most preferably up to about 0.50hours, thereby increasing the adhesion strength and dissolutionresistance of said wound dressing, as previously described above.

The processed wound dressing can then be subjected to a sterilizationstep. The dressing can be sterilized by a number of methods. Forexample, a preferred method is by irradiation, such as by gammairradiation, which can further enhance the blood dissolution resistance,the tensile properties and the adhesion properties of the wounddressing. The irradiation can be conducted at a level of at least about5 kGy, more preferably a least about 10 kGy, and most preferably atleast about 15 kGy. The sterilized wound dressing can be subsequentlypackaged for storage in a heat sealed pouch purged with an inert gassuch as either argon or nitrogen gas.

A wound dressing is produced from said chitosan biomaterial which iscapable of substantially stanching the flow of severe life-threateningbleeding from a wound by adhering the wound dressing to the wound site.The wound dressing is preferably sealed to said wound and prevents bleedout from said wound site by adhering said wound dressing to said woundsite employing clotting and agglutinating of the severe bleeding. Thiswound dressing preferably adheres strongly to the wound site, whileclotting and agglutinating red blood cells from around the wound, sothat pressure need only be employed preferably in the first five minutesof application. In one form of this invention, the device is designed tobe a temporary dressing which is applied, even by unskilledpractitioners, in order to keep the wounded person alive until expertmedical intervention is possible.

In certain applications, the dissolution rate of the subject wounddressing has been relatively slow compared to the agglutination rate,and this balance has produced good results (agglutination at high enoughrate stops dissolution). Also it has demonstrated the importance ofuniformity of the internal and surface structure of the wound dressing.If a substantial defect is present in the wound dressing, such as achannel caused by grain boundaries or minor cracking, then significantblood flow will channel its way along the defect and produce a highlyundesirable bleed-through condition which can flush away the smallerless-viscous agglutination areas as they form. Also significant bloodflow at pressure over the wafer surface appears to adversely affectwound adhesion of prior art wound dressing, but not the wound adhesionof the wound dressing of this invention.

An important preferred attribute of this wound dressing herein is themeans of combining the chitosan with the blood while achieving goodmechanical integrity of the resultant “clot” and good binding of theclot to the surface immediately adjacent to the injury. The subjectwound dressing preferably accelerates blood clot formation at the woundsite, to reinforce clot formation at the wound site and prevent bleedout from the wound site, and to substantially prohibit the flow of bloodand other fluids into and/or out of the wound site.

The wound dressing of the present invention maintains it's extraordinarydual capability for clotting and adhesion to a wound site, as describedabove, while at the same time exhibiting a high level of resilience inan extreme environment. The exceptional resilience of this wounddressing is exemplified by the formidable physical properties thereofwhich are described herein. The subject wound dressing, unlike prior artproducts described above, also has an outstanding ability to conform towound shape while maintaining structural resilience. This structuralresilience is a capacity for the wound dressing to assume a preferredshape after deformation without any substantial loss of mechanicalproperties.

The subject wound dressing, unlike prior art product described above,also has excellent structural memory. Structural memory comprehends thecapacity of the wound dressing to substantially restore its previousshape after deformation.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Photo-digital image of transverse cross section through earlyuncompressed wound dressing.

FIG. 2. Photo-digital image of transverse cross section through orientedlamella structures in uncompressed wound dressing.

FIG. 3. Light photomicrograph of interconnected porous chitosan wounddressing structure sectioned normal to base

FIG. 4. Photograph of chitosan biomaterial wound dressing after heatingand compression.

FIG. 5. Scanning electron photomicrographs of a typical base surface ofcompressed chitosan wound dressing. Higher magnification inset (bar=100micron).

FIG. 6. Histological stained section through chitosan/spleen injury siteand adjacent splenic surface. Agglutinated clot response (A) with amixture of fibrin/platelet rich blood clot (B) between patch and spleen.Very good adhesion between is spleen and chitosan

FIG. 7. Photograph of thoracic aorta injury sealed with chitosan patch

FIG. 8. Fixed thoracic aorta demonstrating perforation injury

FIG. 9. Stained histological section through thoracic aorta injury

FIG. 10. Photograph of in vitro burst pressure failure in a stronglyadherent dressing

FIG. 11. Histogram of water and blood adsorption results for gammairradiated and un-irradiated (unsterilized) samples.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Table 1 provides a list of the main chitosan materials acquired forhemorrhage control testing. With the exception of the Gelfoam™+thrombin,and Surgicel™ controls for swine spleen experiments and the Johnson andJohnson 4″×4″ gauze control for use in swine aortic perforations, thedressing materials were all chitosan-based.

Aqueous solutions (2.00% w/w) were prepared in clean, sterile, 1 literPyrex flasks from Ametek UF water and dry chitosan. In the case of theCarbomer, Primex and Genis chitosan materials, 1.0% or 2.0% w/w ofglacial acetic acid (Aldrich 99.99%) was added to the aqueous mixtures.Dissolution was achieved by shaking of the flask at 40° C. for 12 to 48hours. The solutions were degassed by application of vacuum at 500 mTorrat room temperature immediately prior to freezing.

Wound dressings were prepared from the 2% aqueous solutions of chitosanthat were poured into Teflon™-coated aluminum or polystyrene molds to atleast 1.5 cm deep and frozen in a −80° C. Revco freezer at −45° C. for 3hours. Alternatively, freezing was carried out on the shelves inside aVirtis Genesis 35EL freeze drier. There was at most 10% shrinkage in thewound dressings and the final freeze-dried wound dressing density wasnear 0.033 g/cm³. Transverse cross sections of two types of molded wounddressings are shown in FIGS. 1 & 2 (different freezing rates). Thestructures observed (see also FIG. 3) were affected by the rates ofcooling in the bulk solution and at the different surfaces.Subsequently, structures in the wound dressings were controlled byformulation, mold (size & shape) and freezing conditions. Optimal wounddressing structures were those that were open-porous consisting ofuniform interconnected pores of close to 50 microns in diameter orlamella and hexagonal structures normal to the plane of cooling, Thesestructures could be controlled, yielding flexible yet strong wounddressings of large specific surface areas for highly efficient and rapidblood coagulation. Typically the available specific surface area forsuch structures were greater than 500 cm²/g. The scanning electronphotomicrograph in FIG. 5 shows the typical open cell structure in thebase surface of a wound dressing. The wound dressings were heated in aconvection oven at 80±1° C. for one half hour to optimize the structureand distribution of acetic acid concentration. It was found that thisstep was essential to optimize the adhesive properties of the wounddressing in a bleeding field (typically adhesion to dermis>40 kPa).

The wound dressings were immediately compressed from 17 mm thickness to5.5±0.5 mm at 80±5° C. under a loading of close to 50 kPa. (from ca.density 0.03±0.005 g/cm³ to 0.12±0.02 g/cm³). FIG. 4 shows theappearance of the base of a typical preferred chitosan wound dressingfor hemorrhage control after heating and compression.

A preferred method preparation of hemostatic wound dressings is asfollows:

-   a) Dry chitosan powder or flake with degree of deactylation above    85%, less than 26 ppm metallic component and greater than 90% dry    solids content was made into a 2% aqueous solution (w/w) with 2 or    1% acetic acid (w/w) at 40° C.-   b) The solution of chitosan from a) above was degassed under reduced    pressure at up to 500 mTorr under agitation for at least 5 minutes    and poured into a mold to a depth of 1.7 cm. Certain low-density,    foam structures exhibited problems due to their ready dissolution in    a bleeding field. These problems were generally avoided by thorough    degassing of the solution.-   c) The mould containing the degassed chitosan solution was frozen by    cooling from room temperature to −45° C. A linear cooling ramp was    used over a 90 minute period, and the temperature was maintained at    −45° C. for at least another hour.-   d) The frozen chitosan was then freeze dried using a condenser which    was at a temperature below −70° C. and a vacuum at about 100 mTorr.    The shelf temperature was ramped from −45° C. to −15° C. and held at    that level for 10 hours. A further 36-48 hours of freeze drying at    10° C. was then performed. Freeze drying was performed until    achieving close to about 2.8% of the original frozen plaque mass.-   e) At 2.8% of original mass, the process was stopped and the freeze    dried wound dressing removed from the mold.-   f) The product formed was an acid buffered, water soluble, high    specific surface area wound dressing that had shrunk 10% from its    original frozen volume. The wound dressing structure was generally a    uniform open porous structure with 50 to 80 micron diameter    interconnecting pores. Using a slightly different cooling regime in    which super-cooling was not affected, a lamella/hexagonal structure    (with uniformly thin chitosan sheets close to 5 microns thick with    close to 50 microns separation between sheets) was achieved.-   g) The wound dressing was then compressed (from 1.7 cm to ca. 0.5 cm    thick) between smooth and flat platens heated to 80±2° C. under    application of 60±20 kPa pressure.-   h) Next, the dressing was conditioned in a convection oven by    heating at 80±5° C. for 30 minutes.-   i) Each wound dressing was then stored in labeled Kapak 530 heat    sealed pouches.-   j) The resultant pressed wound dressing was tough, flexible,    hemostatic, adherent to wet tissue and resistant to dissolution by    streaming blood.-   k) Improved dissolution properties, improved adhesion strength and    sterilization were achieved by exposure of the wound dressing to 15    kGy gamma irradiation under nitrogen atmosphere.

TABLE 1 Carbomer Genis Source Protosan (Norway) (USA) Primex (Norway)(Iceland) Sample Name G213 G113 CL213 CL113 9012-75-4 Chitoclear BatchNumber 511-583-01 005-370-01 607-783-02 310-490- VA-UY992 BN 381 TM 752TM 751 SO11115-1 01 Bio-Source Crab &/or Crab &/or Crab &/or Crab ShrimpShrimp Shrimp Shrimp Shrimp Shrimp Shrimp Shrimp &/or Shrimp AppearanceFine white Fine white Fine white Fine Yellowed Fine off- Fine off- Fineoff- Off-white powder powder powder white powder + white white whiteflake powder specks powder powder powder Viscosity cps 108 12 133 12 NANA 109 156 1216 (1% soln) % Dry Matter  93.0 90.3  90.8 93.9 NA NA  97.7 97.6  93 % Protein  0.2  0.2  0.1  0.1 NA NA  <0.3%  <0.3%  <0.3% %Deacetylation  86 85  84 87 90 89  93  91  90 Low Metals* Yes Yes YesYes Yes Yes Yes Yes Yes Salt Glutamate Glutamate Chloride Chloride Nonsalt Non salt Non salt Non salt Non salt *Below accepted limits of lead,mercury, bismuth, antimony, tin, cadmium, silver, copper and molybdenum.NA = not available.

In vivo evaluation of hemostasis of candidate hemorrhage controldressings of varying composition and structure was screened inincreasingly challenging animal models of hemorrhage as hereinafterdescribed. A spleen laceration model was utilized in order to be able toscreen large numbers of candidate dressings in a simple reproduciblemodel and to compare them to conventional materials. Although this isthe least challenging bleeding model (mild oozing bleeding ca. 2-5ml/min), most initial wound dressing formulations failed this test. Alsoall chitosan gels, powders failed in this test while films performedpoorly.

Prior to testing in a severe hemorrhage model, swine were anticoagulatedwith systemic intravenous heparin and better materials were tested in acapsulated spleen stripping model (strong oozing bleeding ca. 10-20ml/min). Those few materials that passed this test were then evaluatedin the carotid laceration model (ca. 50 ml/min) in anticoagulated swine.Wound dressing formulations of candidate materials passing this testwere then tested on the swine aortotomy model with in which 4 mmdiameter perforations in were made in the thoracic or abdominal aortas.Materials passing these challenging models of severe vascular hemorrhage(bleeding rates in excess of 100 ml/min) were also tested in a severe(Grade V) model of hepatic trauma.

The testing described here was carried out on healthy animals that hadpreviously undergone procedures and were scheduled to be sacrificed forevaluation. All experiments were performed in accordance with the 1996Nation Research Council, “Guide for the Care and Use of LaboratoryAnimal” and applicable Federal regulations. After identification of theanimal, anesthesia was induced with Telazol 4-9 mg/kg I/M. Isofluranewas given by mask and the animal was intubated.

The chitosan patches for the laceration and capsular strippingexperiments were either equal size quarter pieces cut from a 37 mmdiameter wound dressing or 1.5 cm×1.5 cm wound dressing pieces cut froma larger wound dressing.

Control materials of Gelfoam™+thrombin or Surgicel™ were prepared from1.5 cm×1.5 cm pieces. Gelfoam™ size 100, absorbable gelatin wounddressing, was supplied by Pharmacia. Oxidized cellulose, Surgicel™, wassupplied by Ethicon. Topical thrombin (bovine origin) 10,000 U.S. unitswas supplied by Jones Pharma. The Gelfoam™+thrombin was prepared beforeuse by soaking of 1.5 cm×1.5 cm×0.8 cm wound dressings in the thrombinfor 30 minutes.

A midline ventral laporatomy was performed. The top half of the spleenwas exteriorized (apposing the surgical wound with towel clamps). Thesurface was kept moist by the application of sterile saline solutionfrom a wet lap pad.

For anticoagulation, the right femoral artery was surgically isolatedand cannulated with a 6F sheath, allowing for collecting blood samples.The activated clotting time (ACT) was measured before administration of5000 units of heparin intravenously, 10 minutes after administration ofheparin and every 20 minutes thereafter. If the ACT level was less than200 seconds, 2000 units of heparin were given and the ACT was remeasuredafter 10 minutes. This was repeated until the ACT>200 seconds to ensurethat the animal was anticoagulated.

The area of splenic testing was demarcated and kept moist by using thetowel clamps and wet pads and only exposing the most immediate untestedsurface.

A single injury was made prior to the application of a test patch, asfollows:

-   -   (i) In the laceration model, the injury (8 mm long×4 mm deep)        was made using a #11 surgical blade positioned in a right-angled        forceps so that 4 mm of blade was protruding.    -   (ii) In the capsular stripping model, the injury (5 mm×5 mm×4 mm        deep) was made using the clamped #11 blade and a pair of        surgical scissors.        After making the injury, bleeding was allowed for 30 seconds.        The surface blood was removed with gauze, following which a test        patch was applied digitally to the injury using a constant        uniform pressure for 30 seconds. The digital pressure was then        removed and the patch was observed for two minutes. At this        stage, the trial number was recorded. If observable rebleeding        occured, the time to rebleed was recorded and the next trial (30        second bleed, clean away blood with gauze, 30 seconds digital        pressure followed by up to 2 minutes observation) commenced. The        trial for a test patch was complete when no rebleeding occurred        in the 2 minute observation period or if 6 trial rebleeds were        observed. If the wound continued to rebleed for 6 trial periods,        then the failed patch was removed and a Gelfoam+thrombin patch        applied. A new injury was made and another patch tested.

In the case of the carotid laceration model, chitosan patches (37 mm×25mm) were cut from the 37 mm diameter compressed wound dressing or largerwound dressings. For facility in application, some of the wounddressings had a top layer of 3M 9781 foam medical tape attached to thechitosan with 3M 9942 skin adhesive. Gelfoam™+thrombin was used as acontrol.

A vertical incision was made exposing a 10 cm length of carotid artery.The fascia was retracted and the surrounding soft tissue was dissecteduntil the artery was supported on a flat base of tissue. Tie-off sutureswere placed proximal and distal to the exposed artery. These wereclamped and a 1.5 cm incision was made longitudinally in the artery.

For anticoagulation, the right femoral artery was surgically isolatedand cannulated with a 6F sheath, allowing for collecting blood samples.The activated clotting time (ACT) was measured before administration of5000 units of heparin intravenously, 10 minutes after administration ofheparin and every 20 minutes thereafter. If the ACT level was less than200 seconds, 2000 units of heparin were given and the ACT was remeasuredafter 10 minutes. This was repeated until the ACT>200 seconds to ensurethat the animal was anticoagulated.

After making the incision, the artery was allowed to bleed for 2 secondsand then was compressed for 1 minute. The compression was removed andthe ties were re-clamped. The area was flushed with saline. The tieswere unclamped 2 seconds before application of a patch. Pressure wasapplied uniformly over the patch for 3 minutes. If bleeding was observedwithin 30 minutes after application of pressure, then another 3 minutesof pressure was re-applied. If the patch was not adhering then it wasreplaced with a new patch. Each re-application of pressure, orreplacement of a patch of the same type were treated as trial periodsfor that patch type. A trial for a particular wound dressing wasconsidered complete if no bleeding was observed from around, or throughthe patch in a 30 minute period. A material was rated on the number oftrials it took to achieve 30 minutes of hemostasis (no observablebleeding from the wound).

In the case of swine aorta perforation, sample patches of compressedchitosan wound dressing cut to 2.5 cm diameter pieces or controls of4″×4″ surgical gauze were used.

Either or both the abdominal and the thoracic aortas were exposed bymidline ventral laporatomies in the former and sternotomies in thelatter. The fascia and sternum were clamped and ties were placedproximal and distal to the sites of incision. While the tie-off clampswere applied, a #11 scalpel blade was used to make a 3 mm incisionthrough the wall of the aorta and a 4 mm diameter Medtronic™ vascularpunch was inserted through the incision to make a 4 mm diameter hole inthe aorta. The punch was removed and the tie-off clamps released withdigital pressure applied to the hole.

The patch was held between thumb and forefinger with the middle fingerapplying pressure to the hole in the aorta. The pressure from thismiddle finger was released for 1 second before application of the wounddressing to the bleeding field. The wound dressing was held in place byfirm pressure applied through the forefinger to the patch over theaortic hole. The pooled blood that escaped the wound during applicationof the patch was suctioned away. After 3 minutes of digital pressure,the finger was removed and the patch observed for any sign of continuedbleeding and poor adherence.

If continued bleeding or re-bleeding was observed in the first 30minutes after application of the patch, then a further 3 minutes ofpressure was applied. If hemostasis was still not complete, then anotherpatch of the same wound dressing was prepared, the old patch removed anda new trial commenced. A trial was considered complete if no bleedingwas observed from around or through the patch in a 30-minute period. Amaterial was rated on the number of trials it took to achieve 30 minutesof hemostasis (no observable bleeding from the wound). Control samplesof gauze were applied in the same manner as the chitosan wound dressingduring a trial.

All animals were euthanized while under anesthesia with an injection ofbarbiturates (Euthasol, 1 ml/10 lb) via an auricular vein. Animals wereeuthanized at the end of the experimental procedure or prior to the endif the animal experienced any untoward effects.

Tests were ranked from 0.0 to 6.0 according to the number of trialsnecessary before hemorrhage control was achieved and the time to rebleed(only in the case of the spleen trials). A test in which only one trialwas necessary and there was no rebleed was ranked as 0.0. A test whichrequired a second trial and the time to rebleed of the first was 90seconds was ranked:

${1.0 + \frac{120 - 90}{120}} = 1.25$(in the case of a spleen) or 1.0 in the other models.

A test which needed four trials to achieve hemostasis and where the timeto splenic rebleed in the third trial was 30 seconds was ranked:

${3.0 + \frac{120 - 30}{120}} = 3.75$(in the case of a spleen) or 3.0 in the other models.

A sample which failed completely by rapid dissolution, lack of adherenceor uncontrolled bleeding was ranked 6.0+.

In summary, the worse the hemostasis, the higher the ranking as definedby the following:

      R = Γ + Λ Where  Γ = number  of  trials  to  stop  bleeding − 1$\mspace{76mu}{\Lambda = \frac{A - {{time}\mspace{14mu}{to}\mspace{14mu}{{rebleed}(s)}\mspace{14mu}{in}\mspace{14mu}{previous}\mspace{14mu}{trial}}}{A}}$     A = time  of  trial(s)

The results of the spleen studies are summarized in Tables 2, 3 and 4.

Table 2 shows the behavior of chitosan test samples that werenon-optimized with respect to composition and structure. Thesenon-optimized materials ranged from, worse to the Surgicel™ negativecontrol (Table 4), to comparable and to only partially better. Thepresence of phosphate buffer solution produced a poorly adherent, slowlyhemostatic patch which was only slightly more effective than Surgicel™.The chitosan film was moderately adherent, providing a reasonable sealto bleeding, however it was only very slowly hemostatic as evidenced bythe slow welling of blood beneath its transparent surface. The earliertrials generally showed signs of a low density foam in the top surfaceof the molded wound dressing. It was found that this low density foamwas susceptible to dissolution and collapse if the top surface of thewound dressing was applied to a bleeding field. It was subsequentlydiscovered that this foam effect could be avoided by degassing ofsolutions before freezing. Low molecular weight chitosan wound dressings(relative 1% solution viscosity<50 cps) were found to be verysusceptible to dissolution in a bleeding field making them unsuitablefor the patch application. The glutamate counter anion produced softerwound dressings but at the cost of producing wound dressings that werereadily dissolved in a severely bleeding field. Low density wounddressings (those less than 0.05 g/cm³) with acetate counterions werealso found to be readily compromised by dissolution and collapse.

Table 3 shows the result rankings of the optimized chitosan wounddressings of preferred composition and structure. These wound dressingswere composed of chitosan with higher molecular weights (relative 1%solution viscosity greater than 100 cps) and had wound dressingdensities close to 0.12 g/cm³. In the moderately bleeding spleen tests,the results for the optimized wound dressings were found, using aWilcoxon Rank Sum W Test, to be indistinguishable from the positivecontrol of Gelfoam™+thrombin (Z=−0.527, p=0.598). Using the samestatistical method, the wound dressings were shown to be significantlydifferent from the poorly performed Surgicel™ control (Z=−3.96,p=0.0001).

FIG. 6 demonstrates (via a H&E stained histological section) the closeadherence of the optimized chitosan wound dressings patches to thespleen surface as well as the agglutination of erythrocytes at theimmediate vicinity of the injury.

The rankings for the carotid injury model are summarized in Table 5. Inthis model, the optimized chitosan patch performed very well in trials3, 5 and 6. The improvement in performance over the first trials 1 and 2was due to the application of the support backing (3M 9781 foam bandage)to the immediate top surface of the wound dressing. This backing enabledmore uniform pressure to be applied over the wound dressing and allowedfor the person applying the dressing to remove their fingers easily fromthe patch surface without them sticking and inducing patch detachmentfrom the wound. The carotid model was used to investigate more severearterial bleeding conditions than were possible in the spleen injurymodel. Gelfoam™+thrombin was investigated as a possible positive controlbut was found to dissolve in a highly bleeding field.

Table 6 summarizes the results of the aortic injury model. Gauze bandage(4″×4″) was used as a control bandage. It was found that the control wasunable to stop severe bleeding in all trial periods whereas theoptimized chitosan aortic patches were able quickly to stop andsubsequently clot the very high level of bleeding observed in this woundafter only 1 or 2 applications of the patch. The exact significance(two-tailed p=0.002) was determined for the probability that there wasno difference between rankings of sample and control. On average theblood loss after patch application was minimal (<50 ml) if the wound wasstanched on the first attempt. If a second attempt was required bloodloss after patch application was greater than 100 ml but less than 300ml. On average less than 150 ml of blood was lost after patchapplication in the case of the chitosan wound dressing while, in thecase of the 3 gauze control studies, more than 1 liter of blood was lostfor each animal. In the case of the chitosan wound dressing study,survival was 100%, while in the case of the gauze study, none (0%) ofthe animals survived. The chitosan patches demonstrated continuedhemostatic efficacy over the trial period of 30 minutes and until theanimals were sacrificed which was generally 1 to 2 hours later. FIG. 7demonstrates a typical chitosan patch sealing a severe thoracic wound.The lumen side (showing the injury) of the resected aorta sealed by thepatch in FIG. 7 is shown in FIG. 8. FIG. 9 shows a photomicrograph of astained histological section taken through the injury of FIGS. 7 & 8.Evidence of strong clotting at the injury site was found on removal andinspection of aortas on animal sacrifice (FIG. 9) and, in the case oftrial number 16, where after dislodging a patch in a live animal (aftermore than 30 minutes of application) there was no subsequentre-bleeding.

TABLE 2 Sample Sample Sample Sample Sample Model Model Result ResultAnimal Type Source Name Batch Form Anticoagulat. Injury Rank Comments1346 Chitosan Carbomer 9012-75-4 VA-UY992 PBS treat No Laceration 4.0Poor adhesion + slow hemostasis 1346 Chitosan Carbomer 9012-75-4VA-UY992 Film type No Laceration 2.7 Adhesive + slow hemostatic 1345Chitosan Carbomer 9012-75-4 VA-UY992 PBS treat No Laceration 5.6 PoorAdhesion + poor hemostasis 1338 Chitosan Primex Chitoclear TM 752 Densetype Yes Laceration 3.1 Slow bleed through low density defect in wounddressing 1338 Chitosan Pronova G113 005-370-01 Dense type Yes Capsular6.0+ Low Mw Chitosan dissolved 1338 Chitosan Pronova G113 005-370-01Dense type Yes Capsular 6.0+ Low Mw Chitosan dissolved 1338 ChitosanPronova G213 511-583-01 Dense type Yes Capsular 6.0 Softer ChitosanCollapsed 1338 Chitosan Pronova G213 511-583-01 Dense type Yes Capsular6.0 Softer Chitosan Collapsed 1441 Chitosan Primex Chitoclear BN 381 LDtype, Yes Capsular 1.8 Good adhesion + initial small FF type bleedthrough 1441 Chitosan Primex Chitoclear BN 381 Dense type, Yes Capsular1.8 Good adhesion + initial small bleed FF type Dense type = Dense wounddressing (ca. 0.12 g/cm³) PBS treat = A wound dressing neutralized bysoaking in phosphate buffer saline solution LD type = Low density wounddressing (ca. 0.03 g/cm³) FF type = A fast frozen wound dressing Filmtype = a solvent cast film (500 microns)

TABLE 3 Sample Sample Sample Sample Sample Model Model Model ResultResult Animal Type Source Name Batch Form Anticoagulat. Organ InjuryRank Comments 1346 Chitosan Carbomer 9012-75-4 VA-UY992 Dense type NoSpleen Laceration 0.0 Good adhesion + rapid hemostasis 1404 ChitosanPrimex Chitoclear BN 381 Dense type No Spleen Laceration 0.0 Goodadhesion + rapid hemostasis 1404 Chitosan Carbomer 9012-75-4 VA-UY992Dense type No Spleen Laceration 0.0 Good adhesion + rapid hemostasis1404 Chitosan Primex Chitoclear BN 381 Dense type Yes Spleen Capsular0.0 Good adhesion + rapid hemostasis 1338 Chitosan Primex Chitoclear TM752 Dense type Yes Spleen Laceration 0.0 Good adhesion + rapidhemostasis 1338 Chitosan Primex Chitoclear TM 752 Dense type Yes SpleenLaceration 0.0 Good adhesion + rapid hemostasis 1338 Chitosan PrimexChitoclear TM 752 Dense type Yes Spleen Capsular 0.0 Good adhesion +rapid hemostasis 1338 Chitosan Primex Chitoclear TM 752 Dense type YesSpleen Capsular 0.0 Good adhesion + rapid hemostasis 1338 ChitosanPrimex Chitoclear TM 752 Dense type Yes Spleen Capsular 0.0 Goodadhesion + rapid hemostasis 1338 Chitosan Primex Chitoclear TM 752 Densetype Yes Spleen Capsular 0.0 Good adhesion + rapid hemostasis 1344Chitosan Pronova CL213 607-783-02 Dense type Yes Spleen Capsular 0.0Good adhesion + rapid hemostasis 1344 Chitosan Primex Chitoclear TM 752Dense type Yes Spleen Capsular 0.0 Good adhesion + rapid hemostasis 1441Chitosan Primex Chitoclear TM 752 Dense type Yes Spleen Capsular 0.0Good adhesion + rapid hemostasis 1441 Chitosan Primex Chitoclear TM 752Dense type Yes Spleen Capsular 0.0 Good adhesion + rapid hemostasis 1441Chitosan Primex Chitoclear TM 752 Dense type Yes Spleen Capsular 0.0Good adhesion + rapid hemostasis 1441 Chitosan Primex Chitoclear TM 752Dense type Yes Spleen Capsular 1.3 Good adhesion + initial slight localbleeding 1478 Chitosan Primex Chitoclear TM 751 Dense type Yes SpleenCapsular 0.0 Good adhesion + rapid hemostasis 1478 Chitosan PrimexChitoclear TM 751 Dense type Yes Spleen Capsular 0.0 Mean 0.1 ±0.31Dense type = Dense wound dressing (ca. 0.12 g/cm³)

TABLE 4 Sample Sample Sample Sample Model Model Result Animal TypeSource Name Form Anticoagulat. Injury Result Rank Comments 1338 ControlPharmacia Gelfoam + thr Sponge Yes Capsular 0.0 Satisfactory adhesion +rapid hemostasis 1344 Control Pharmacia Gelfoam + thr Sponge YesCapsular 0.0 Satisfactory adhesion + rapid hemostasis 1344 ControlPharmacia Gelfoam + thr Sponge Yes Capsular 0.0 Satisfactory adhesion +rapid hemostasis 1441 Control Pharmacia Gelfoam + thr Sponge YesCapsular 0.0 Satisfactory adhesion + rapid hemostasis 1441 ControlPharmacia Gelfoam + thr Sponge Yes Capsular 0.0 Satisfactory adhesion +rapid hemostasis Mean 0.0 ±0.0 1338 Control Ethicon Surgicel Gauze YesLaceration 5.9 Very slow hemostasis 1441 Control Ethicon Surgicel GauzeYes Capsular 3.9 Very slow hemostasis and poor adhesion 1441 ControlEthicon Surgicel Gauze Yes Capsular 5.9 Very slow hemostasis and pooradhesion Mean 5.2 ±1.15

TABLE 5 Trial Sample Sample Sample Sample Sample Model Model ResultResult Number Animal Type Source Name Batch Form Anticoagulat. InjuryRank Comments 1 1404 Chitosan Primex Chitoclear BN 381 Dense type YesLaceration 3 First trial injury. Profuse bleeding at site prior toapplication, first 3 applications slowed bleeding final applicationclosed wound area not initially covered. 2 1335 Chitosan PrimexChitoclear TM 752 Dense type Yes Laceration 4 Non-backed sample. Problemwith pressure application without damaging wound dressing in bleedingfield. 3 1335 Chitosan Pronova CL213 607-783- Dense type Yes Laceration0 3M backing enabled pressure 02 application without damaging wounddressing. 5 1442 Chitosan Primex Chitoclear TM 752 Dense type YesLaceration 0 3M backing + Good adhesion & rapid hemostasis. 6 1442Chitosan Primex Chitoclear TM 752 Dense type Yes Laceration 1 3Mbacking + Good adhesion & rapid hemostasis. 4 1441 Control PharmaciaGelfoam + Sponge Yes Laceration  6+ Unable to stop bleeding th asGelfoam dissolved in bleeding field and was non-adherent Dense type =dense sponge wound dressing (ca. 0.12 g/cm³)

TABLE 6 Trial Sample Sample Sample Sample Model Result Number AnimalType source Name Sample Batch Form Anticoagulat. Aorta Model Injury RankResult Comments 1 1465 Chitosan Primex Chitoclear TM 752 Dense type NoAbdom. 4 mm perfor. No backing. Sealed for 4 mins then bled through. 21465 Chitosan Primex Chitoclear TM 752 Dense type No Abdom. 4 mm perfor.0 3M backed. Sandwich around Aorta. Hemostatic & adherent after 30 mins.3 1470 Chitosan Primex Chitoclear TM 752 Dense type No Thor. 4 mmperfor. 0 3M backed. Remaining hemostatic & adherent after 30 mins. 41468 Chitosan Primex Chitoclear TM 752 Dense type No Thor. 4 mm perfor.0 3M backed. Remaining hemostatic & adherent after 30 mins. 5 1462Chitosan Primex Chitoclear TM 752 Dense type No Thor. 4 mm perfor. 1 3Mbacked. First patch Temporarily adherent. Second Patch remaininghemostatic & adherent after 30 mins. 6 1460 Chitosan Primex ChitoclearTM 752 Dense type No Thor. 4 mm perfor. 0 3M backed. Remaininghemostatic & adherent after 30 mins. 7 1460 Chitosan Primex ChitoclearTM 752 Dense type No Abdom. 4 mm perfor. 1 3M backed. First patchslipped off. Second Patch remaining hemostatic & adherent after 30 mins.8 1461 Chitosan Primex Chitoclear TM 752 Dense type No Thor. 4 mmperfor. 0 3M backed. Remaining hemostatic & adherent after 30 mins. 91461 Chitosan Primex Chitoclear TM 752 Dense type No Abdom. 4 mm perfor.0 3M backed. Remaining hemostatic & adherent after 30 mins. 10 1469Chitosan Primex Chitoclear TM 752 Dense type No Thor. 4 mm perfor. 3 3Mbacked. First 3 patches slipped off. Fourth patch remained hemostatic &adherent after 30 mins 11 1469 Chitosan Primex Chitoclear TM 752 Densetype No Abdom. 4 mm perfor. 0 3M backed. Sandwich necessary as aortaperforated above and bel 12 1467 Chitosan Primex Chitoclear TM 751 Densetype No Abdom. 4 mm perfor. 1 3M backed. First Patch removed afterbleeding through side in first 5 mins. Second patch remained adherent &hemostatic over tr 13 1467 Chitosan Primex Chitoclear TM 751 Dense typeNo Abdom. 4 mm perfor. 1 3M backed. First Patch removed after collapsingin first 5 minutes Second patch remained adherent & hemostatic overtrial 14 1398 Chitosan Primex Chitoclear TM 751 Dense type No Abdom. 4mm perfor. 0 3M backed. Remaining hemostatic & adherent after 30 mins.15 1398 Chitosan Primex Chitoclear TM 751 Dense type No Thor. 4 mmperfor. 1 3M backed. First patch slipped off. Second Patch remaininghemostatic & adherent after 30 mins. 16 1399 Chitosan Primex ChitoclearTM 751 Dense type No Abdom. 4 mm perfor. 0 3M backed. Remaininghemostatic & adherent after 30 mins. Knocked off while making secondnearby injury after 40 minutes. Injury had completely clotted over. 171399 Chitosan Primex Chitoclear TM 751 Dense type No Abdom. 4 mm perfor.2 3M backed. First 2 patches slipped off. Third patch remainedhemostatic & adherent after 30 mins. 18 1479 Chitosan Primex ChitoclearTM 751 Dense type No Abdom. 4 mm perfor. 1 3M backed. First patchslipped off. Second Patch remaining hemostatic & adherent after 30 mins.19 1479 Chitosan Primex Chitoclear TM 751 Dense type No Thor. 4 mmperfor. 2 3M backed. First 2 patches slipped off. Third patch remainedhemostatic & adherent after 30 mins. Mean   0.7 ±0.89 20 1483 ControlJ&J Gauze No Abdom. 4 mm perfor.  6+ After 15 minutes animal becamehypotensive and was euthanized 21 1489 Control J&J Gauze No Abdom 4 mmperfor.  6+ After 15 minutes animal became hypotensive and waseuthanized 22 1490 Control J&J Gauze No Thor 4 mm perfor.  6+ After 15minutes animal became hypotensive and was euthanized Dense type = densesponge wound dressing (ca. 0.12 g/cm³)

Preferably, the hemorrhage control dressing described above includes asurface, which grips the wound area to substantially avoid slipping ofthe dressing during use. Typically, this non-slip surface of thedressing comprises a traction surface. The subject hemorrhage controldressing may benefit from having an effective non-slip surface, such asa traction surface. The subject hemorrhage control dressing can have asmooth and rough side. The rougher side would preferably be the tissueor bleeding surface side if that side also demonstrated better adhesiveproperties.

A traction surface may improve a dressing ability to control rapidarterial bleeding by providing increased stability of surface contact(better traction) on a well lubricated surface (such as those surfaceswhich present in the case of severe bleeding). Such a traction surfacewould help to channel blood, without adversely affecting adhesionkinetics while allowing for a more controlled and stable tissue contactduring the critical period of dressing application. For example, thetissue side of the bandage could have a traction surface in the form ofa tread design. This could prevent the dressing from undergoing tractionloss in a direction away from the wound when undergoing application tothe wound.

The non-slip surface of the hemorrhage control dressing could beproduced with ridges that are non-connecting or blinded to one another.Thus, in turn, the channels formed between the ridges would be fully orpartially blinded to one another and thus provide a controlledconnection that would provide for a controlled blood flow back into orout of the wound area. The controlled blood flow in area of dressingapplication could be maintained by the ridges or specific types ofresponsive gates in the hemorrhage control dressing. Ridges on bottom ofa mold for producing the hemorrhage control dressing may includedepressions of the type which will permit a non-slip surface, forexample, in the form of traction controls such as ridges or the like, inthe subject dressings.

A hemorrhage control dressing could therefore be produced having atleast one non-slip surface, such as a traction surface. Also, a methodof producing such a dressing could be provided. Finally, a mold to aproduce a hemorrhage control dressing, as described above, can befabricated.

So as to treat severe hemorrhage in cases where adhesive base and topsurfaces are advantageous, it is possible to design the support backingso that if necessary it could be readily peeled away when adhesion andclotting are required on both surfaces.

There are numerous hemorrhage control configurations of the dressingdescribed above to address a wide range of possible types of hemorrhagicwound. It is envisioned there be will a need to be able to carry (in abattlefield situation) several bandages of differing configurations sothat the injured persons can be treated by the first responder or evenpotentially by injured persons themselves. The dressing of the inventionclaim is robust and can tolerate a great deal of physical abuse andstill remain an active hemorrhage control platform.

The dressing is ideal for treating focal vascular bleeding and smalltopical wounds. It is also well suited to packing into complex entrywounds where the bleeding site cannot be easily compressed.

Once hemorrhage control is achieved with the current invention,stabilizing an extremity wound, approximating wound edges and creating adurable dressing that will prevent contamination and allow evacuation ofthe injured for definitive repair are the main requirements for acivilian and a battlefield hemorrhage control dressing.

One envisioned configuration of the hemorrhage control dressing is a10″×18″ dressing with a flexible, elastic backing that can be tightlyattached around an extremity and secured with a locking tab such as apermanent adhesive glue via a peel back surface to itself. Such a deviceconfiguration would approximate wound surfaces and add a hemorrhagecontrol surface without compromising blood flow to the distal extremity.Such a dressing could be applied by a first responder or in someinstances by the injured soldier and would be stable under ambulation orextremity movement during transport. It is envisioned that the bandagewould be removed by cutting it apart with no adverse adhesion to thewound or skin.

The US Army Science and Technology Objective (STO) A, HemorrhageControl, was established in 2000 to advance the need for hemorrhagecontrol on the battlefield. The general strategic objective of the STOcan be summarized as the development of products and methods that willreduce the number of deaths due to hemorrhage in battlefield casualties.The requirements for hemorrhage control products and methods were statedthus:

They must be practicable for use by one or more of the following: self(wounded combatant), buddy (fellow non-medical soldier who aids thewounded soldier), combat lifesaver, combat medic, physician assistant,and battalion surgeon. They must be practicable for use in far forwardfield conditions including rugged terrain, limited visibility, andenvironmental extremes. Products and methods must not require externalelectrical sources. All devices must be man-portable and durable. It isexpected that products and methods that are useable far forward willalso be used at higher echelons of care. A specific strategic objectiveof the STO is the development of new or improved hemostatic agents foruse on compressible hemorrhage under far forward field conditions. Asingle product for use on compressible and non-compressible sites isdesired.

As part of STO, a study of hepatic hemorrhage control in a swine livermodel was conducted at the US-Army Institute of Surgical Research (ISR)at Fort Sam Houston, San Antonio, Tex. using the hemorrhage controlbandage of this invention. The study was conducted to determine theeffect of the chitosan hemorrhage control bandage on blood loss andsurvival in a standardized model of severe venous hemorrhage and hepaticinjury in swine. This model has been used to study numerous otherhemostatic bandages at US-Army ISR.

Cross-bred commercial swine were used in this study. Animals weremaintained in a facility accredited by the Association for theAssessment and Accreditation of Laboratory Animal Care, International.This study was approved by the Institutional Animal Care and UseCommittee of the US Army Institute of Surgical Research, Fort SamHouston, Tex. Animals received humane care in accordance with the Guidefor the Care and Use of Laboratory Animals (National Institutes ofHealth publication 86-23, revised 1996).

Animals were assigned randomly to receive either the Chitosan bandagesor Gauze sponges (see Table 7). Surgical preparation consisted of thefollowing: Animals were fasted 36-48 hours prior to the surgicalprocedure, with water allowed ad libidum. After premedication withglycopyrrolate and a combination of tiletamine HCl and zolazepam HCl(Telazol®, Fort Dodge Laboratories, Fort Dodge, Iowa), anesthesia wasinduced by mask using 5% isoflurane. The swine were intubated, placed ona ventilator, and maintained with isoflurane. Carotid arterial andjugular venous catheters were placed surgically. Laparotomy wasperformed and splenectomy and urinary bladder catheter placement werecompleted. A rectal temperature between 37.0° and 39.0° C., and 15minutes of stable mean arterial pressures (MAP) were required prior tofurther experimental procedures. Blood pressure and heart rate wererecorded at 10-second intervals throughout the study period using acontinuous data collection system (Micro-Med®, Louisville, Ky.).Baseline arterial blood samples were collected from each animal toconfirm that each animal exhibited normal platelet count, prothrombintime, activated partial thromboplastin time, and plasma fibrinogenconcentration.

Liver injuries were induced as previously reported. The method includedthe following. The liver was retracted by manually elevating the leftand right medial lobes to allow adequate exposure. Next, a speciallydesigned clamp with two 4.5 cm sharpened tines configured in the form ofan ‘X’ was positioned with the center approximately 2-3 cm dorsal to theintersection of the left and right medial lobes, on the diaphragmaticsurface of the liver. The base plate of the instrument was positionedbeneath the quadrate lobe, on the visceral surface. The injury wasinduced by clamping the tines of the instrument through the parenchymaand underlying vessels of the two medial lobes so that the tines wereseated in corresponding grooves in the base plate of the instrument.After the first penetration of the liver, the instrument was opened andthe tines were withdrawn and repositioned to the animals left such thatthe second application would overlap the first by 50 percent. Followingthis repositioning, the liver was penetrated a second time.Documentation of the liver injury was achieved by excision andinspection of the liver at the conclusion of the experimental period.The injuries appeared as large stellate wounds with a small island oftissue in the center, and measured approximately 10×8×4 cm. The injurieswere through and through, with one or more of the left medial lobarvein, right medial lobar vein, and portal hepatic vein lacerated.

Thirty seconds after injury, resuscitation was initiated with warm (38°C.) lactated Ringer's solution in all animals. The goal of resuscitationwas return to baseline MAP. Fluid was administered at 260 ml/min. Thisresuscitation regimen was continued until the goal was reached andreinitiated if MAP decreased, throughout the 60 minute study period.Simultaneously with initiation of resuscitation (30 secondspost-injury), treatments were applied as follows. One dressing wasapplied to the surface of the quadrate lobe to cover the penetratinginjury and two other dressings were stuffed into the injury from thediaphragmatic aspect. Compression was applied for 60 seconds in thedorso-ventral direction. After 60 seconds, the injury was inspected todetermine whether hemostasis was achieved. Next, the applicator's handswere repositioned and pressure was applied for 60 seconds in thelatero-medial direction, and the observation for hemostasis wasperformed. This sequence was repeated for a total of four 60 secondcompressions. If hemostasis was complete after any compression, nofurther compressions were performed. Hemostasis was defined as theabsence of visually detectable bleeding from the injury site.

Following completion of treatment application, the abdomen was closedand the animal was monitored for 60 minutes after injury or until death,whichever came first. Death prior to 60 minutes was defined as a heartrate of 0. At 60 minutes, surviving animals were euthanized by anoverdose of pentobarbital.

Immediately after induction of the injury, blood was continuouslysuctioned from the peritoneal cavity until the start of treatmentapplication. The volume was determined and designated as pre-treatmentblood loss. At the end of the study period, each abdomen was opened andthe liquid and clotted intra-peritoneal blood were suctioned andmeasured. This was designated as post-treatment blood loss.Additionally, total resuscitation fluid use was recorded. Preinjuryanimal blood volume was estimated using the equation: estimated bloodvolume (ml)=161.4751(body weight^(−0.2197))(body weight), as we havepreviously reported (Pusateri, 2001).

Body weight, estimated blood volume, number of vessels lacerated,baseline MAP, survival time, preinjury MAP, pretreatment blood loss, andbandage adherence scores were analyzed by analysis of variance using theGLM procedure of SAS. Data are reported as least squares mean±standarderror of least squares mean. Data were examined for heterogeneity ofvariance and non-normality. These conditions were detected forpost-treatment blood loss and fluid use data. Therefore, blood loss andfluid use data were log transformed prior to analysis. The transformeddata were analyzed by analysis of variance. These data are expressed asback transformed means and 95% confidence interval (95% CI).Distribution of females and males, hemostasis, and survival data wereanalyzed by Fishers Exact Test using the FREQ procedure of SAS. Data arereported as proportions or percentages. Two sided tests were used forall comparisons.

There were no differences among treatment groups in animal body weight,estimated blood volume, distribution of animal sexes, baseline MAP,preinjury MAP, number of major vessels lacerated within the liverinjury, or pretreatment blood loss (See Tables 8 and 9).

Post-treatment blood loss was reduced in the Chitosan group, compared tothe Gauze wound dressing control (p=0.01). No significant difference influid use was observed. Survival percentage was increased in theChitosan group (p=0.04). Hemostasis occurred more frequently in theChitosan group at 3 and 4 minutes post-injury (p=0.03). Survival timescould not be statistically compared because of the high level ofsurvival in the Chitosan group (See Table 10).

TABLE 7 Test Material Lot Number And Related Information Gauze DressingJohnson and Johnson NU Gauze Sponge (Negative Control) General Use 10.2cm × 10.2 cm. Rayon/Polyester Formed Fabric. Lot Number 1999-051399T5205B2. Chitosan Wound dressing Oregon Medical Laser Center,Chitosan Wound dressing, 10.2 cm × 10.2 cm. Lot Number 052101. BatchNumber E1041. 26 May 2001 (1% AA, Primex Lot # 751).

TABLE 8 Gauze Sponge P value of Variable Control Group Chitosan Groupdifference n 7 8 N/A Body Weight (kg) 39.1 ± 1.2 38.7 ± 1.1 0.82Estimated Blood Volume 2819 ± 66  2800 ± 64  0.83 (ml) Female/Male (n/n)5/2 6/2 0.88 Baseline MAP (mm Hg) 71.3 ± 3.6 68.8 ± 3.3 0.50 PreinjuryMAP 69.1 ± 4.8 69.3 ± 4.4 0.98 (mm Hg) Hematocrit (%) 32.2 ± 1.1 32.6 ±1.0 0.79 Hemoglobin (g/dL) 11.2 ± 0.4 11.3 ± 0.3 0.80 Platelets(1000/ul) 567 ± 28 502 ± 25 0.11 PT (sec) 10.7 ± 0.2 10.6 ± 0.2 0.70aPTT (sec) 15.7 ± 0.9 16.5 ± 0.9 0.56 Fibrinogen (g/dL)  159 ± 0.9 180 ±8  0.10

TABLE 9 Gauze Sponge Chitosan P value of Variable Control Group Groupdifference Number of Vessels Lacerated  1.86 ± 0.29  1.88 ± 0.27 0.96Pretreatment Blood Loss (ml) 296.1 ± 55.4 291.1 ± 55.4 0.95 PretreatmentBlood Loss  10.6 ± 2.0  10.3 ± 2.0 0.94 (ml/kg body weight)

TABLE 10 Gauze Sponge P value of Variable Control Group Chitosan Groupdifference Post-treatment Blood Loss (ml) 2879 (788-10,513; 264 (82-852;95% <0.01 95% CI) CI) Post-treatment Blood Loss 102.4 (28.2-371.8) 9.4(2.9-30.3; <0.01 (ml/kg body weight) 95% CI) Fluid Use (ml) 6614(2,519-17,363; 1793 (749-4,291; 0.03 95% CI) 95% CI) Survival (%) 28.687.5 0.04 Survival Time (min; 38.4 ± 5.8 (n = 5) 10.0 (n = 1) N/Anonsurvivors only) Hemostasis at 1 Minute (%)  0 50 0.08 Hemostasis at 2Minutes (%)  0 50 0.08 Hemostasis at 3 Minutes (%)  0 62 0.03 Hemostasisat 4 Minutes (%)  0 62 0.03

This US-Army ISR study (Pusateri et al 2002) demonstrates, in anindependent study, the significantly improved performance of thechitosan wound dressing over standard 4″×4″ gauze. The US-Army ISR hasonly been able to demonstrate significantly improved performance over4″×4″ gauze in the stanching of severe blood flow in the case of thedressing of this invention claim and in the case of a dry FibrinThrombin wound dressing being developed by the Red Cross. The Red CrossBandage is costly, as well as being delicate and prone to breakage.

High molecular weight 4″×4″ chitosan hemorrhage control dressings with3M 9781 porous foam backing have been prepared from an Icelandic shrimpsource (Genis Lot#SO1115-1). These were prepared with 2% acetic acid and2% chitosan solution using a commercial freeze drying company to preparea large sterile lot of chitosan bandages (Lot#OMLC_(—)2SM114). Thebandages were irradiated at 15 kGy under nitrogen. They weresubsequently tested for uniaxial tensile strength, burst strength, bloodadsorption, water adsorption as well as for sterility. Swine aortaperforations were carried out on non-gamma irradiated samples inabdominal and thoracic injuries. Seven patches were used. On averageblood loss after patch application was <50 ml. All patches wereadherent, wound sealing and hemostatic on their first application (7×0rankings). All animals survived.

Both gamma-irradiated and un-irradiated bandages (Lot#OMLC_(—)2SM114)were tested with an in vitro burst pressure test developed at OregonMedical Laser Center in Portland Oreg. To perform a burst test, a 25 mmdiameter circular test piece of the bandage is immersed in citratedwhole blood for 10 seconds. The test piece is then placed centrallyover, and firmly held with digital pressure, on a 4 mm diameterperforation in the side of a 50 mm diameter PVC pipe for 3 minutes.After this initial attachment, fluid pressure inside the pipe is rampedat 4.5±0.5 kPa.s⁻¹, with pressure and time recorded at 0.1 secondintervals. Burst pressure is recorded as the maximum pressure recordedprior to failure. An adhesive failure ranking is assigned to assess therelative adhesiveness of the bandage to the test site. The rankingsystem is separated into 3 distinct modes of failure. A ranking of 1 isgiven to a test piece which is readily separated from the PVC surfacewith no chitosan remaining adhered. A ranking of 2 is assigned when thetest piece is less readily detached and some of the chitosan remainsattached to the test site. A ranking 3 is assigned when the test piececan only be removed by cohesive separation of the bulk wound dressingfrom the base structure which remains firmly fixed to the PVC surface.

The average burst pressure of gamma irradiated and un-irradiatedchitosan bandages (Mean±SD, n=6) on a PVC substrate using blood aswetting medium was 122±1.9 kPa and 86±20 kPa, respectively. The resultswere analyzed statistically using a T-test (p=0.007). The averageadhesive failure rankigs of gamma irradiated and un-irradiated chitosanalpha bandages (Mean±SD, n=6) on a PVC substrate using blood as wettingmedium were both 3±0. FIG. 10 shows an image of a high ranking failurewhere cohesive failure has occurred within the chitosan structure.

The blood and water adsorption properties of the dressings (Lot#OMLC_(—)2SM114) were determined by immersing small test pieces (ca. 0.02g) in blood or water for 3.0 seconds. Difference in mass before andafter immersion was recorded.

The average mass of medium adsorbed in 3 seconds per one gram of wounddressing was determined for gamma irradiated and un-irradiated chitosansamples (n=4) using blood or water as the wetting medium (see FIG. 11).The results were analyzed statistically using a one-way ANOVA with aTukey-HSD test, p=0.001. Gamma irradiation significantly reduced theexcessive adsorption of water in the case of the non-irradiatedmaterial. Such excessive water adsorption would cause wound dressingcollapse (into a gel) with subsequent adhesive and structural failure.

Tensile test pieces of the chitosan dressings (Lot#OMLC_(—)2SM114) wereevaluated using a uniaxial Chatillon Materials Testing Vitrodyne V1000equipped with a 5 kg load cell. Samples were cut into dog-bone pieces(15±1 mm×6.5±0.5 mm×5±0.5 mm gauge×thickness×width) and held between twoclamps. The crosshead speed was 10 mm.s⁻¹. Load and displacement wererecorded at 0.1 second intervals.

Tensile results are shown in Table 11. There were no significantdifferences between gamma irradiated and un-irradiated samples withrespect to both stress and strain. There was a small increase in Youngsmodulus with irradiation at 15 kGy.

TABLE 11 Youngs Modulus Ultimate Load* Ultimate Sample (Mpa) (kg)Elongation No irradiation 1.8 ± 1.2 2.2 ± 0.2 0.9 ± 0.1 (n = 5) Gammairradiation 4.4 ± 2.7 2.3 ± 0.2 0.85 ± 0.15 (n = 9) *Calculated for a2.5 cm wide bandageFifty two 4″×4″ chitosan wound dressings (Lot#OMLC_(—)2SM114) wereprepared cleanly. Of these 4″×4″ wound dressings, 46 were packaged in adouble pack envelope and were sent to the IsoMedix facility in Ontario,Calif. for irradiation with gamma radiation at a certified dose between14-15 kGy. Boxed with these samples were a set of 8 staphylococus aureus(ATCC 29213) doped chitosan wound dressing bars (1″×0.21″×0.21″) cutfrom wound dressing 2SM114#1. Each bar was inoculated with 100microliters of 0.5 MacFarlane inoculum. The staphylococcus aureus wasswabbed from a demonstrably active control culture. A control set of 4bars with no staphylococus was also included. Control samples with nogamma radiation treatment were kept in small sterile containers in heatsealed envelopes at room temperature and in the dark (see Table 12 for asummary of the controls).

TABLE 12 Lot # Control # Pieces Dosed Gamma OMLC_2SM114 61 4 No NoOMLC_2SM114 55 4 No No OMLC_2SM114 65 4 Yes No OMLC_2SM114 77 4 Yes NoOMLC_2SM114 74 4 No Yes OMLC_2SM114 71 4 Yes Yes OMLC_2SM114 58 4 YesYes

The 46 irradiated wound dressing packages were opened under sterileconditions with sterile handling, an ethylene oxide sterile adhesivecoated foam backing (3M 9781 tape) was attached, a small off-cut piece(ca. 1.2″×0.2×0.12″) of each wound dressing and backing was removed forindividual wound dressing sterilization testing and the wound dressingswere repackaged inside the original inner pack by heat sealing. 40 ofthese wound dressings were labeled with lot number and wound dressingnumber and sent out for evaluation. The off-cut and control pieces weregiven to the microbiology facility at St Vincent's PHS for sterilitytesting.

The off-cut pieces and control pieces were placed aseptically in labeledsample vessels (0.6″ diam.×5″) containing enriched thioglycolate growthmedia and incubated aerobically at 35° C. The culture media wereexamined at 7, 14 and 21 days for indications of growth. The sampleswere subcultured in TSA W/5% sheep's blood, incubated at 35° C. andexamined for growth after 48 hours.

The individual cultures were analyzed by turbidity testing andsubculture swabbing. Absence of any growth in all the cultures and allthe subcultures at 7, 14 and 21 days was demonstrated, even thosecultures which were un-irradiated and dosed with staphylococcus aureus.Gram positive staining of particular cultures collaborated thesefindings.

The invention claimed is:
 1. A method for producing a chitosan wounddressing comprising providing an aqueous solution including a chitosanbiomaterial; placing the aqueous solution in a mold; freezing theaqueous solution within the mold by cooling the mold and aqueoussolution according to prescribed conditions to form a frozen chitosanstructure within the mold; removing water from the frozen chitosanstructure by a prescribed freeze-drying process to form a spongechitosan structure having a thickness and a density; compressing thesponge chitosan structure by the application of heat and pressure toreduce the thickness and increase the density of the sponge chitosanstructure to form a densified chitosan structure; and preconditioningthe densified chitosan structure by heating the densified chitosanstructure according to prescribed conditions to form a wound dressinghaving adhesion strength and resistance to dissolution in high bloodflow bleeding situations.
 2. A method according to claim 1, furtherincluding degassing the aqueous solution before freezing the aqueoussolution.
 3. A method according to claim 1, wherein the application ofheat and pressure during the compression comprises a compressiontemperature that is not less than about 60° C.
 4. A method according toclaim 1, wherein the prescribed conditions of preconditioning of thedensified chitosan structure comprises heating the densified chitosanstructure to a temperature of up to at least about 75° C.
 5. A methodaccording to claim 1, wherein the prescribed conditions ofpreconditioning of the densified chitosan structure comprises a periodof time up to at least about 0.25 hours.
 6. A method according to claim1, wherein the application of heat and pressure during the compressioncomprises a compression temperature that is between about 60° C. toabout 85° C.
 7. A method according to claim 1, wherein the prescribedconditions of preconditioning of the densified chitosan structurecomprises heating the densified chitosan structure to a temperature thatis up to about 85° C.
 8. A method according to claim 1, wherein theprescribed conditions of preconditioning of the densified chitosanstructure comprises a period of time up to about 0.5 hours.